The extent to which intestinal stem cells (ISCs) are targeted and depleted during immune-mediated gastro- intestinal (Gl) damage and the role of the immune system in regulating ISC-mediated regeneration are poorly understood. Likewise, while T cells are known to migrate to the intestines, their specific localization within the tissue and proximity to the ISC compartment when mediating disease are largely unknown. In collaboration with the NIDDK-sponsored Intestinal Stem Cell Consortium, this proposal assembles a multi-disciplinary team to elucidate how the immune system can influence the ISC compartment during intestinal damage and regeneration, as well as to evaluate approaches to overcome such damage and promote recovery of injured epithelium. Our preliminary findings indicate that ISCs and their ability to mediate regeneration are lost during both alloreactive injury from graft vs. host disease (GVHD) and autoreactive injury from regulatory T cell depletion, and that co-cultures of immune cells and gut organoids can be used to model and mechanistically dissect these processes. We have also found that Interleukin-22 (IL-22) is an important signal from the immune system augmenting ISC-mediated regeneration after injury. New preliminary data developed in response to reviewer comments indicate that our autoimmunity model mirrors GVHD with acute activation of effector T cells and upregulation of GI homing molecules, infiltration of the GI tract with activated T cells, and reduced epithelial damage after IL-22 treatment. F-652, a novel rhIL-22 dimer developed by Generon Corp., has translational potential to promote ISC recovery and epithelial regeneration in vivo. Additional new data demonstrate that our 3-D imaging approach can quantify loss of ISCs, specific invasion of the ISC compartment, and localization of vascular integrin ligands in GVHD. New mechanistic data indicate that MAdCAM-1 blockade specifically inhibits T cell crypt invasion and protects ISCs, indicating this pathway to be an important regulator of ISC injury, and not just overall gut migration. We will test the hypotheses that 1) T cell recruitment to the ISC compartment, depletion of ISCs, and loss of their regenerative capacity are common features of immune-mediated epithelial injury in the GI tract and 2) IL- 22 administration can protect ISCs and promote regeneration after damage. ISC function and depletion will be evaluated with autoreactive and alloreactive experimental approaches, utilizing in vivo mouse models of systemic autoimmunity and GVHD as well as ex vivo cultures of mouse and human intestinal organoids in collaboration with UMC Utrecht, their organoid biobank, and their advanced ISC expertise. IL-22 treatment will be evaluated as an epithelial-targeted regenerative immunotherapy promoting ISC function. No therapy exists currently to accelerate GI recovery from immunopathology. This project will lead to a mechanistic understanding of fundamental interactions between the ISC compartment and the immune system, opening a new direction for treatment of inflammatory GI diseases by protecting ISCs and augmenting regeneration.